Currently a major problem with the ES cell-based cell replacement therapies is cellular heterogeneity (Conley et al., Fetal Diagn Ther 19: 218-23, 2004). The generation of homogeneous cell populations, which are not available in sufficient quantities from fetal or adult neural tissues, is a prerequisite for clinical use. Typically, the neuronal cultures derived from mouse or human ES cells contain a variety of neuronal subtypes as well as non-neural cells. Recently, stroma-produced inductive signals and transcription factors were used to enrich for the subtypes of neurons, particularly dopaminergic neurons and motor neurons. For example, stromal co-cultures (Kawasaki et al., Neuron 28: 31-40, 2000) and transcription factors such as Nurr1 (Chung et al., Eur J Neurosci 16: 1829-38, 2002; Kim et al., Nature 418: 50-6, 2002) increase the generation of dopaminergic neurons. Soluble ligands, such as sonic hedgehog, increase the generation or motor neurons capable of integration into the host tissue after transplantation {Wichterle, 2002 #1933}. However, even in the best conditions, only a small percent of human ES cell-derived midbrain dopamine neurons are electrophysiologically active (Perrier et al., Proc Natl Acad Sci USA 101: 12543-8, 2004). The prior art cell separation techniques use magnetic beads (U.S. Pat. No. 6,833,269; Carpenter) differential digestion of rosettes (U.S. Patent Application No. 20030068819; Zhang et al.), or promoter-based isolation of neural stem cells (U.S. Patent Application No. 20040029269, Goldman et al.).
The transplantation of human Neural Precursor Cells (hNPCs) has been shown to mitigate various neurodegenerative disorders (Flax et al., Nat. Biotechnol. 16: 1033-1039, 1998; Tamaki et al., J Neurosci Res 69: 976-86, 2002; Lindvall et al., Nat Med 10 Suppl: S42-50, 2004). The primary human tissue is limiting and the long term in vitro expansion of hNPCs compromises their multilineage potential, migration, and differentiation capacity after transplantation (Cadwell and Joyce PCR Methods Appl. 2: 28-33, 1992; Schwartz et al., J Neurosci Res 74: 838-51, 2003). On the other hand, human Embryonic Stem (hES) cells (Thomson et al., Science 282: 1145-7, 1998) represent virtually unlimited source of a variety of human cell types. Several group reported that neural precursor cells (e.g., rosettes) can be found among other cells in differentiated HESC cultures can give rise to neural precursors (Carpenter et al., Exp Neurol 172: 383-97, 2001; Reubinoff et al., Nat Biotechnol 19: 1134-40, 2001; Zhang et al., Nat Biotechnol 19: 1129-33, 2001). All current protocols, however, use the initial formation of heterogeneous embryoid bodies and require differential enzymatic digestion (Zhang et al., Nat Biotechnol 19: 1129-33, 2001), extensive passaging (Reubinoff et al., Nat Biotechnol 19: 1134-40, 2001), and/or immuno-enrichments (Carpenter et al., Exp Neurol 172: 383-97, 2001). Controllable differentiation procedure resulting in homogenous target population is the key for rigorous analysis of cellular and molecular changes during hESCs to hNPCs transition and a uniform population of transplantable hNPCs is mandatory for tissue therapies in clinics (Snyder et al., Adv. Neurol. 72: 121-132, 1997; Odorico et al., Stem Cells 19: 193-204, 2001; Hornstein and Benvenisty, J Neurosci Res 76: 169-73, 2004).
The importance of controlled efficient differentiation of human ESC has been underscored by generation of human definitive endoderm (D'Amour et al., Nat Biotechnol 23: 1534-41, 2005) and generation of pure Pax-6-positive mouse neuronal precursors from mouse ES cells (Bibel et al., Nat Neurosci 7: 1003-9, 2004; Plachta et al., Development 131: 5449-56, 2004). Giving substantial differences between mouse and human ES cells (Ginis et al., Dev Biol 269: 360-80, 2004; Daheron et al., Stem Cells 22: 770-8, 2004; Humphrey et al., Stem Cells 22: 522-30, 2004) it is important to find out if clinically relevant human ESCs can be efficiently differentiated to homogeneous hNPCs in defined culture conditions.
Thus there is a need in the art for a method for generating large populations of highly purified, homogeneous neural precursor cells.